Abstract
Cry1 protoxins of Bacillus thuringiensis are insecticidal 135-kDa proteins synthesized and assembled into parasporal crystals during sporulation. After ingestion, these crystals dissolve in the midgut and active toxins with molecular masses of about 65-kDa are released from the N-terminal half of the molecule by midgut proteases. Direct synthesis of the toxin-containing N-terminal half of Cry1 molecules using recombinant DNA techniques results in a low level of unstable truncated proteins that do not crystallize. In the present study, inclusions of truncated Cry1C (Cry1C-t) were obtained by combining genetic elements from other endotoxin genes and operons that enhance Cry protein synthesis and crystallization. Increased levels of Cry1C-t synthesis were achieved by using cyt1A promoters to drive expression of the 5′ half of cry1C that included in the construct the 5′ cry3A STAB-SD mRNA stabilizing sequence and the 3′ stem-loop transcription terminator. RNA dot blot analysis showed that the STAB-SD and 3′ transcriptional termination sequences were important for stabilization of truncated cry1C (cry1C-t) mRNA. A low level of cry1C-t mRNA was present when only the cyt1A promoters were used to express cry1C-t, but no accumulation of Cry1C-t was detected in Western blots. The orientation of the transcription terminator was important to enhancing Cry1C-t synthesis. Inclusion of the 20- and 29-kDa helper protein genes in cry1C-t constructs further enhanced synthesis. The Cry1C-t protein was toxic to Spodoptera exigua larvae, though the toxicity (50% lethal concentration [LC50] = 13.2 μg/ml) was lower than that of full-length Cry1C (LC50 = 1.8 μg/ml). However, transformation of the HD1 isolate of B. thuringiensis subsp. kurstaki with the cry1C-t construct enhanced its toxicity to S. exigua as much as fourfold.
Insecticidal Cry proteins produced by Bacillus thuringiensis are the principal active ingredients of most bacterial insecticides. Based on mass, there are two major types of Cry proteins, those with molecular masses of approximately 135 kDa, such as the common Cry1 protoxins, and those with molecular masses of approximately 70 kDa, exemplified by Cry2A, Cry3A, and Cry11A (16). The amino acid sequence of the latter type corresponds to the amino acid sequence of the N-terminal half of the former type. Cry proteins typically are synthesized as protoxins during sporulation and are assembled into crystals that stabilize the toxin (3, 8). When ingested by insects, the crystals dissolve in the midgut and the protoxin is cleaved by midgut proteases, releasing an active polypeptide with a molecular mass of 65 to 68 kDa (1, 16, 17).
Because the C-terminal half of 135-kDa Cry1 protoxins is not toxic, if it could be eliminated and the cellular resources could be redirected to synthesize an equivalent additional amount of the N-terminal half, the specific toxicity—i.e., the toxicity per unit of mass of bacterial insecticides—might be improved. This would in essence convert Cry1 proteins by truncation into toxins like Cry2A or Cry3A. When truncated cry1 genes are expressed in B. thuringiensis, however, the toxin yields are low, and the truncated proteins do not form inclusions (1, 15, 22). Possible reasons for this include a low level of truncated gene expression, instability of the truncated mRNA and protein, and inefficient crystallization of the truncated protein.
Several genetic elements that enhance synthesis and crystallization of “naturally truncated” Cry toxins, such as Cry2A, Cry3A, and Cry11A, have been identified recently. These elements include the 5′ STAB-SD mRNA stabilizing sequence of cry3A (2), 3′ stem-loop structures that also stabilize cry transcripts (24), and two helper proteins that enhance translation and/or crystallization. These two helper proteins are the 20-kDa chaperone-like protein encoded by orf3 of the cry11A operon (6, 25, 26) and the 29-kDa protein encoded by orf2 of the cry2A operon (23), which apparently serves both as a molecular chaperone (5) and a scaffolding protein that facilitates Cry2A crystal formation (7).
Several studies have shown that these elements, alone or in combination with each other or other genetic elements, can be manipulated to enhance Cry synthesis. For example, Park et al. (13) demonstrated that Cry3A synthesis could be increased more than 10-fold in comparison to the synthesis by the wild-type strain by driving expression of cry3A, including the STAB-SD sequence, with strong cyt1A promoters. In another study, synthesis of Cry2A and Cry11A using the cyt1A promoters–STAB-SD expression system resulted in more moderate but still significant increases of 4.4- and 1.3-fold, respectively (14). With respect to the helper proteins, the 20-kDa protein enhances net synthesis of Cry2A (7).
In the present study, we made various combinations of these enhancer elements and evaluated them to determine their capacities to enhance the synthesis and crystallization of truncated Cry1C (Cry1C-t) molecules. Here we show that inclusions of Cry1C-t can be produced by combining mRNA stabilizing sequences and helper protein genes in constructs containing truncated cry1C (cry1C-t). We also show that adding this construct to a wild-type strain of B. thuringiensis improves its insecticidal activity against the beet armyworm, Spodoptera exigua, an important insect pest.
MATERIALS AND METHODS
Bacterial strains and transformation.
Plasmid constructs were amplified in Escherichia coli DH5α. The cry1C constructs were expressed in an acrystalliferous strain of B. thuringiensis subsp. israelensis (4Q7), B. thuringiensis subsp. kurstaki HD-1, or B. thuringiensis subsp. aizawai 1857. The B. thuringiensis strains were transformed by electroporation as previously described (13).
PCR.
The PCR was performed by using the Expand Long Template PCR system (Boehringer, Mannheim, Germany) or with Vent (Exo+) DNA polymerase (New England Biolabs).
Construction of cry1C-t.
The plasmid constructs used are shown in Fig. 1 and Table 1, and the primers used for gene amplification are listed in Table 2. Plasmid p2-44, which contained the intact cry1C gene (GenBank accession number X96682), was provided by Abbott Laboratories (North Chicago, Ill.). The cry1C gene was obtained as a 4.8-kb EcoRI-HindIII fragment from p2-44, filled with the Klenow fragment, and cloned into the SmaI site of pHT3101 (11) to generate pPF1C. The cry1C open reading frame (ORF) in pPF1C was amplified with primers 1Ca-1 and 1Ca-3 (Table 2) by using a DNA thermal cycler (Perkin Elmer GeneAmp PCR system 2400) and inserted into expression vector pPF-CH that contained the cyt1A promoters and STAB-SD sequence to generate pPFT1Cs. The cry1C-t fragment was amplified with primers 1Ca-1 and 1Ca-2 and inserted into pHTCytA (13), which contained cyt1A promoters, to generate pPFT1C-t or into pHTCytA with STAB-SD (pPF-CH) to generate pPFT1Cs-t. The cry1C-t gene encodes 630 amino acids, and the C-terminal amino acid is K630. The 479-bp fragment containing the transcription termination sequence (TTS) in pPFT3As (13) was obtained by PCR using primers 3Aa-1 and 3Aa-2. The 479-bp fragment was digested with SphI and cloned into the same site in pPFT1Cs-t to generate pPFT1Cs-t3(+), with the TTS in the same orientation as the cry1C ORF, and pPFT1Cs-3t(−), with the TTS in the orientation opposite that of the cry1C ORF. The 1.5-kb fragment in pPFT11Ast containing the 20-kDa gene was amplified with primers 11Aa-1 and 11Aa-2 and inserted into the SphI site of pPFT1Cs-t to generate pPFT1Cs-20k. The 850-bp fragment in pPFT2Asf containing orf2 of the cry2A operon was amplified with primers 2Aa-1 and 2Aa-2 and inserted into the XbaI-SalI site of pPF-CH to generate pPF-ORF2. The 2.38- and 3.41-kb SalI-SphI fragments were obtained from pPFT1Cs-3t(+) and pPFT1Cs-20k, respectively, and were inserted into the SalI-SphI site of pPF-ORF2 to generate pPFT1Csf-3t(+) and pPFT1Csf-20k.
FIG. 1.
Summary of vector construction for expression of full-length and truncated cry1C genes. (A) The vectors pPFT3As and pPF1C were used as templates for further construction. The full-length cry1C gene, including its promoter region, was obtained as a 4.8-kb fragment of plasmid 2-44 (see Materials and Methods) partially digested with EcoRI and HindIII. This fragment was treated with the Klenow fragment and inserted into pHT3101 to generate pPF1C. (B) The full-length and truncated cry1C ORFs beginning at the ATG codon were inserted into the SalI-SphI sites and SalI sites of pPF-CH (see Materials and Methods) to generate pPFT1Cs and pPFT1Cs-t, respectively. For cry1C-t without the STAB-SD sequence, the same fragment used for construction of pPFT1Cs-t was inserted into SalI sites of pHTCytA. To add the 3′ TTS to truncated cry1C-t, a 479-bp cry3A termination sequence was inserted into the SphI site of pPFT1Cs-t [pPFT1Cs-3t(+) and pPFT1Cs-3t(−)]. (C) The vectors pPFT2Asf and pPFT11Ast were used as templates for amplification of orf2 and the 20-kDa protein gene. (D) For the 20-kDa protein, a 1.5-kb fragment from pPFT11Ast was inserted into the SphI site of pPFT1Cs-t (pPFT1Cs-20k). For the ORF2 protein gene, a 850-bp fragment from pPFT2Asf was inserted into the XbaI-SalI site of pPF-CH (pPF-ORF2). Then 2.38- and 3.41-kb fragments, obtained from SalI-SphI partial digestion of pPFT1Cs-3t(+) and pPFT1Cs-20k, respectively, were inserted into the SalI-SphI site of pPF-ORF2 to generate pPFT1Csf-3t(+) and pPFT1Csf-20k.
TABLE 1.
Summary of genetic elements present in each cry1C construct
| Construct | Genetic elements presenta |
|---|---|
| pPF1C | cry1C p + full-length cry1C |
| pPFT1Cs | cyt1A p + STAB-SD + full-length cry1C |
| pPFT1C-t | cyt1A p + cry1C-t |
| pPFT1Cs-t | cyt1A p + STAB-SD + cry1C-t |
| pPFT1Cs-3t(+) | cyt1A p + STAB-SD + cry1C-t + stem-loop (5′-3′) |
| pPFT1Cs-3t(−) | cyt1A p + STAB-SD + cry1C-t + stem-loop (3′-5′) |
| pPFT1Csf-3t(+) | cyt1A p + STAB-SD + orf2 + cry1C-t + stem-loop (5′-3′) |
| pPFT1Cs-20k | cyt1A p + STAB-SD + cry1C-t + 20-kDa protein gene |
| pPFT1Csf-20k | cyt1A p + STAB-SD + orf2 + cry1C-t + 20-kDa protein gene |
p, promoters (i.e., multiple promoters for all constructs listed).
TABLE 2.
Primers used to amplify cry1C, cry2A, cry3A, and cry11A sequences
| Primer | Sequencea |
|---|---|
| 1Ca-1 | 5′-ACGCGTCGACCGGAGGTATTTTATGGAGGAAAAT-3′ |
| 1Ca-2 | 5′-ACGCGTCGACTTACTTTTGTGCTCTTTCTAAATCAGA-3′ |
| 1Ca-3 | 5′-ACATGCATGCCCCCTTAGATAGATATCATAGAATTG-3′ |
| 3Aa-1 | 5′-ACATGCATGCATTAACTAGAAAGTAAAGAAGTAG-3′ |
| 3Aa-2 | 5′-ACATGCATGCAAGCTTACAGAGAAATACACGAGGG-3′ |
| 2Aa-1 | 5′-GCTCTAGAATAGGAGGAAAAGATTTTATGCTAAAA-3′ |
| 2Aa-2 | 5′-ACGCGTCGACAAATATCTAGTTTTATATTAA-3′ |
| 11Aa-1 | 5′-ACATGCATGCAGTCATGTTAGCACAAGAGGA-3′ |
| 11Aa-2 | 5′-ACATGCATGCTTTAGGTCTTTAAAAATTAGA-3′ |
The ribosome binding site, start codon, and artificial stop codon are indicated by boldface type, and the SalI, SphI, and XbaI restriction enzyme sites are underlined.
cry1C-specific antisense probe.
A 0.8-kb cry1C-specific antisense DNA probe was made by unidirectional PCR using digoxigenin-labeled nucleotides (Boehringer) and the 1Ca-2 primer. The truncated cry1C-t gene PCR product was used as the substrate for Vent (Exo+) DNA polymerase (New England Biolabs).
RNA isolation and dot blot analysis.
RNA was isolated from sporulating cells grown in 10 ml of nutrient broth plus salts (NBG) at 30°C for 12 h (14). The bacterial cells were centrifuged at 6,000 × g for 5 min at 4°C, and the pellets were suspended in 1 ml of TRIzol reagent (GIBCO BRL, Grand Island, N.Y.). Sodium dodecyl sulfate (SDS) was added to a final concentration of 1% (vol/vol). The suspension was sonicated 10 times on ice at 50% duty cycle for 15 s (Ultrasonic Homogenizer 4710 series; Cole-Parmer Instrument Co., Chicago, Ill.). After samples were incubated at room temperature for 5 min, 200 μl of chloroform was added. The samples were mixed thoroughly and centrifuged at 12,000 × g for 15 min. The aqueous phase was transferred to a fresh tube, and 500 μl of isopropanol was added. After incubation at room temperature for 10 min, RNA was collected by centrifugation at 12,000 × g for 10 min. The RNA pellets were washed with 1 ml of 75% ethanol, spun at 7,500 × g for 5 min, dried, and dissolved in 50 μl of diethyl pyrocarbonate-treated double-distilled water. RNA concentrations were determined by measuring the absorption at 260 nm with a PM6 spectrophotometer (Zeiss, Oberkochen, Germany). RNA samples (1 and 2.5 μg) were spotted on a nylon membrane (Micron Separations, Inc., Westborough, Mass.). The membrane was dried in a vacuum oven at 80°C for 2 h. Prehybridization and hybridization at 42°C with the antisense-cry1C DNA probe and detection with the CDP-Star reagent (Boehringer) were performed according to the manufacturer's protocol. After exposure, the detection film was scanned with the GAS 4000 gel documentation system (Evergreen). The level of hybridization was quantified by using ImageQuant 4.1 densitometry software (Molecular Dynamics, Sunnyvale, Calif.). Hybridization values were determined by comparison to the signal obtained with reference plasmid pPFT1C-t, which was assigned a value of 1. RNA dot blots were replicated three times by using three different RNA preparations from three different cultures. Data from the blots were analyzed with the Super ANOVA program (Abacus Concepts, Berkeley, Calif.) (13, 14).
SDS-PAGE.
Bacterial strains were grown in 50 ml of NBG (14) at 30°C for 5 days, by which time the cells had sporulated and lysed. Spores, crystals, and cell debris were pelleted in 1.2-ml aliquots by centrifugation at 15,000 × g for 1 min. The pellets were suspended in 50 μl of 5× sample buffer (10) and boiled for 5 min. After centrifugation at 15,000 × g for 5 min to remove solids, 10-μl samples were loaded onto a 12% polyacrylamide gel and the proteins were separated by electrophoresis. Samples taken from three sets of cultures grown on different days were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE), and the gels were scanned and quantified with the Super ANOVA program (Abacus Concepts) (13, 14).
Purification of Cry1C inclusions.
Sporulated cells were pelleted by centrifugation at 6,500 × g for 15 min, suspended in 15 ml of distilled water, and sonicated twice at 50% duty cycle for 15 s by using the Ultrasonic Homogenizer 4710. Five-milliliter samples were loaded onto a discontinuous NaBr gradient (9), which was then centrifuged at 20,000 × g for 45 min at 10°C in a Beckman L7-55 ultracentrifuge. Bands containing inclusions were collected and dialyzed in water overnight at 4°C. Purified inclusions were pelleted and lyophilized.
Western blot analysis.
Protein concentrations were determined by the method of Bradford (4). Proteins in 5- or 10-μg samples were separated by electrophoresis in an SDS–10% polyacrylamide gel and electroblotted onto a polyvinylidene difluoride membrane (Micron Separations, Inc.) by using a model PS50 electroblotter (Hoefer Scientific Instruments). Western blot analysis was performed by using primary rabbit anti-Cry1C antibody kindly provided by W. J. Moar (Department of Entomology, Auburn University, Auburn, Ala.) and alkaline phosphatase-conjugated goat anti-rabbit immunoglobulin G (Southern Biotechnology Associates, Inc., Birmingham, Ala.) as the secondary antibody (20). Binding of the secondary antibody was detected with the nitroblue tetrazolium and 5-bromo-1-chloro-3-indolyl phosphate (BCIP) reagents (Promega, Madison, Wis.).
Microscopy.
Sporulating cultures were monitored and photographed with a DMRE phase-contrast microscope (Leica) at a magnification of ×1,000.
Bioassays.
Bioassays with neonate S. exigua larvae were carried out as previously described (12). The assays were performed in 24-well plates (Corning) by using lyophilized powder preparations containing spores and crystal inclusions mixed in artificial diet, as described by Moar et al. (12). Two larvae were placed in each well and then held at 28°C under a daily regime consisting of 16 h of light and 8 h of darkness. A total of 48 larvae were used for each protein concentration assayed. Larval mortality was determined after 7 days of exposure to the spore-crystal toxin mixture; larvae were considered to be alive if they were able to respond to tactile stimulation.
RESULTS
Expression of cry1C-t.
When cry1C-t was expressed using pPFT1C-t (Fig. 1), which contained cyt1A promoters to drive expression but lacked the STAB-SD and cry3A 3′ stem-loop sequences, the transcript level as determined by RNA dot blots was low (Fig. 2, lane 1). Inclusion of STAB-SD in the construct (pPFT1Cs-t) yielded a twofold increase in the level of cry1C-t transcript detected (Fig. 2, lane 2). When both STAB-SD and the 3′ stem-loop were included in the construct [pPFT1Cs-3t(+)], the transcript level increased to 2.6 times that of the construct which lacked these elements, pPFT1C-t (Fig. 1; Fig. 2, lane 3; Table 1). Placement of the cry3A stem-loop in the orientation opposite that of cry1C-t [pPFT1Cs-3t(−)] (Fig. 1; Table 1) resulted in a marked reduction in the level of transcript detected (Fig. 2, lane 4).
FIG. 2.
Transcript levels for different cry1C-t constructs. Lane 1, pPFT1C-t (cyt1A p + cry1C-t); lane 2, pPFT1Cs-t (cyt1A p + STAB-SD + cry1C-t); lane 3, pPFT1Cs-3t(+) (cyt1A p + STAB-SD + cry1C-t + stem-loop [5′-3′]); lane 4, pPFT1Cs-3t(−) (cyt1A p + STAB-SD + cry1C-t + stem-loop [3′-5′]); lane 5, pPFT1Csf-3t(+) (cyt1A p + STAB-SD + orf2 + cry1C-t + stem-loop [5′-3′]); lane 6, pPFT1Cs-20k (cyt1A p + STAB-SD + cry1C-t + 20-kDa protein gene); lane 7, pPFT1Csf-20k (cyt1A p + STAB-SD + orf2 + cry1C-t + 20-kDa protein gene). The ratios shown for lanes 2 through 7 are relative to the value for the dot in lane 1, which was assigned a value of 1. Each value represents the average value (ratio) obtained from three separate experiments. Different letters beneath the ratios indicate that values were significantly different at P = 0.05.
No significant differences were found between the cry1C-t transcript levels with constructs that contained the 29-kDa protein genes [pPFT1Csf-3t(+)] and the cry1C-t transcript levels with constructs that contained the 20-kDa protein genes (pPFT1Cs-20k) (Fig. 2, lanes 5 and 6, respectively). These levels were only 62 and 66%, respectively, of those detected with pPFT1Cs-3t(+). However, inclusion of the genes encoding both the 29- and 20-kDa proteins in a construct (pPFT1Csf-20k) (Fig. 1; Table 1) increased the cry1C-t transcript level to a level comparable to that observed with pPFT1Cs-3t(+) (Fig. 2, lanes 3 and 7).
Synthesis of Cry1C-t and inclusion formation.
Constructs which lacked the STAB-SD and cry3A 3′ stem-loop sequences (pPFT1C-t) (Fig. 1) or contained the STAB-SD sequence and cry3A stem-loop in the orientation opposite that of cry1C-t [pPFT1Cs-3t(−)] produced little or no detectable Cry1C-t 68-kDa protein as determined by Western blot analysis (Fig. 3A and B, lanes 1 and 4). No Cry1C-t inclusions were observed in strains transformed with these constructs (Fig. 4A). However, when various elements that stabilized the transcript or enhanced net protein synthesis were included in the constructs, substantial increases in Cry1C-t levels were obtained, and inclusions of this protein were observed (Fig. 3A and B, lanes 2, 3, 5, 6, and 7, and 4B and C). For example, when the STAB-SD sequence was included in pPFT1C-t (pPFT1Cs-t) (Fig. 1; Table 1) or the cry3A stem-loop sequence, placed in the same orientation as cry1C-t, was included in pPFT1Cs-t [pPFT1Cs-3t(+)] (Fig. 1; Table 1), the constructs produced Cry1C-t (Fig. 3A and B, lanes 2 and 3). Comparable levels of Cry1C-t were also detected with constructs that contained the 29- or 20-kDa protein genes (Fig. 3A and B, lanes 5 and 6), though inclusions were observed in only about 33% of the sporulating cells (data not shown). The sizes of these inclusions were similar to those in cells with pPFT1Cs-3t(+). The inclusions produced by the construct containing both the 20- and 29-kDa protein genes (pPFT1Csf-20k) (Fig. 1; Table 1) appeared to be larger than those produced by the other constructs (Fig. 4C). In most cells, two separate inclusions were observed in each cell when the constructs contained either the 20-kDa (pPFT1Cs-20k), 29-kDa [pPFT1Csf-3t(+)], or 20- and 29-kDa (pPFT1Csf-20k) protein genes (Fig. 4C).
FIG. 3.
Synthesis of Cry1C-t by different constructs as determined by SDS-PAGE and Western blot analysis. (A and B) SDS–12% PAGE gel (A) and Western blot of the same gel (B). The relative amounts of Cry1C-t produced by the strains are indicated below the lanes in panel A. Lane 1, pPFT1C-t (cyt1A p + cry1C-t); lane 2, pPFT1Cs-t (cyt1A p + STAB-SD + cry1C-t); lane 3, pPFT1Cs-3t(+) (cyt1A p + STAB-SD + cry1C-t + stem-loop [5′-3′]); lane 4, pPFT1Cs-3t(−) (cyt1A p + STAB-SD + cry1C-t + stem-loop [3′-5′]); lane 5, pPFT1Csf-3t(+) (cyt1A p + STAB-SD + orf2 + cry1C-t + stem-loop [5′-3′]); lane 6, pPFT1Cs-20k (cyt1A p + STAB-SD + cry1C-t + 20-kDa protein gene); lane 7, pPFT1Csf-20k (cyt1A p + STAB-SD + orf2 + cry1C-t + 20-kDa protein gene); lane M, molecular size marker. (C) Control for the Western blot analysis: B. thuringiensis subsp. israelensis, which produces Cry11A and Cyt1A. Lane 1, SDS-PAGE gel; lane 2, Western blot of the same gel. The ratios shown for lanes 3 through 7 are relative to the amount of the 58-kDa protein in lane 2, which was assigned a value of 1. Each value represents the average value (ratio) obtained from three separate experiments. Different letters beneath the ratios indicate that values were significantly different at P = 0.05.
FIG. 4.
Phase-contrast micrographs of sporulated cells of B. thuringiensis subsp. israelensis 4Q7 that expressed representative constructs. (A) pPFT1C-t (cyt1A p + cry1C-t); (B) pPFT1Cs-3t(+) (cyt1A p + STAB-SD + cry1C-t + stem-loop [5′-3′]); (C) pPFT1Csf-20k (cyt1A p + STAB-SD + orf2 + cry1C-t + 20-kDa protein gene). The arrows indicate inclusions formed by Cry1C-t.
A putative 68-kDa band corresponding to Cry1C-t was not observed in protein profiles of strains containing pPFT1Cs-t, pPFT1Cs-3t(+), pPFT1Csf-3t(+), pPFT1Cs-20k, and pPFT1Csf-20k, although at least three novel proteins with molecular masses ranging from 55 to 58 kDa were observed (Fig. 3A, lanes 2, 3, 5, 6, and 7, respectively). Inclusions that were spherical or ovoidal were present in cells transformed with each of these plasmids. The level of Cry1C-t synthesis (Fig. 3A and B) generally corresponded with the cry1C-t transcript levels detected (Fig. 2). For example, constructs which contained the STAB-SD and cry3A stem-loop sequences in the same orientation as cry1C-t [pPFT1Cs-3t(+)] and constructs that contained the 29- and the 20-kDa protein genes along with the STAB-SD sequence (pPFT1Csf-20k) showed the highest levels of cry1C-t transcript and subsequent synthesis of the corresponding protein. The protein yields of the strains containing these constructs were, respectively, 1.4- and 1.7-fold greater than the amount of Cry1C-t produced by pPFT1Cs-t, which lacked the cry3A stem-loop sequence and helper protein genes (i.e., the genes encoding the 29- and 20-kDa proteins) (Fig. 2 and 3B, lanes 3 and 7). Similar levels of transcript and Cry1C-t synthesis occurred in strains transformed with constructs containing either the 29-kDa protein gene [pPFT1Csf-3t(+)] or the 20-kDa protein gene (pPFT1Cs-20k) (Fig. 2 and 3, lanes 5 and 6). These strains produced, respectively, 1.2- and 1.3-fold more Cry1C-t than the strain with pPFT1Cs-t produced.
Although the masses of the 55- to 58-kDa proteins did not correspond to the predicted mass of Cry1C-t (68 kDa), Western blotting with anti-Cry1C antibody confirmed that their bands were composed of Cry1C-t (Fig. 3B, lanes 2, 3, 5, 6, and 7). SDS-PAGE and Western blot analyses of purified inclusions showed that the major component was the 68-kDa Cry1C-t molecule (Fig. 5). Together, these results indicate that there is considerable degradation of Cry1C-t after synthesis (Fig. 3B and 5B).
FIG. 5.
Purified inclusions of Cry1C-t as determined by SDS-PAGE and Western blot analysis. (A) SDS–10% PAGE gel; (B) Western blot of the same gel. Lane 1, molecular size marker; lanes 2 and 3, pPFT1Cs-3t(+) (cyt1A p + STAB-SD + cry1C-t + stem-loop [5′-3′]); lanes 4 and 5, pPFT1Csf-20k (cyt1A p + STAB-SD + orf2 + cry1C-t + 20-kDa protein gene). Five micrograms (lanes 3 and 5) or 10 μg (lanes 2 and 4) of protein was loaded and separated.
Toxicity of Cry1C-t.
The results of bioassays performed with neonate S. exigua larvae are shown in Tables 3 and 4. Two constructs that had the highest levels of Cry1C-t synthesis were compared with the construct containing full-length cry1C by using B. thuringiensis subsp. israelensis 4Q7 as the host strain in each case (Table 3). Neither Cry1C-t construct was nearly as toxic as the full-length molecule. The 50% lethal concentrations (LC50s) for pPFT1Cs-3t(+), which contained the STAB-SD and cry3A stem-loop sequences in the same orientation as cry1C-t, and pPFT1Csf-20k, which contained STAB-SD plus the 29- and 20-kDa protein genes, were 23.4 and 13.2 μg/ml, respectively, whereas pPF1C, which included the cry1C promoter to drive full-length cry1C, was significantly more toxic (LC50 = 1.9 μg/ml).
TABLE 3.
Comparative toxicities to neonate S. exigua of spore and inclusion mixtures of B. thuringiensis strains containing full-length and truncated cry1C constructs
| Straina | LC50 (μg/ml) | Slope ± SEM |
|---|---|---|
| 4Q7/pPF1C | 1.9 (0.2–4.8)b | 0.87 ± 0.25 |
| 4Q7/pPFT1Cs-3t(+) | 23.4 (13.5–37.5) | 1.31 ± 0.22 |
| 4Q7/pPFT1Csf-20k | 13.2 (6.5–22.8) | 1.22 ± 0.21 |
All strains were grown in 50 ml of NBG for 5 days at 30°C and lyophilized.
The values in parentheses are the 95% fiducial limits.
TABLE 4.
Comparative toxicities to neonate S. exigua of spore and inclusion mixtures from commercial B. thuringiensis strains and strains transformed with full-length and truncated cry1C genes
| Straina | LC50 (μg/ml) | Slope ± SEM |
|---|---|---|
| HD-1 (Dipel) | 17.6 (9.7–30.5)b | 1.53 ± 0.30 |
| HD-1/pPFT1Csf-20k | 7.3 (1.1–15.2) | 1.17 ± 0.36 |
| HD-1/pPFT1Cs | 3.5 (1.8–7.4) | 0.92 ± 0.18 |
| 1857 (Xentari) | 2.0 (1.1–3.3) | 1.44 ± 0.24 |
| 1857/pPFT1Csf-20k | 2.0 (1.1–3.5) | 1.19 ± 0.21 |
| 1857/pPFT1Cs | 1.6 (0.8–3.1) | 0.99 ± 0.19 |
All strains were grown in 50 ml of NBG for 5 days at 30°C and lyophilized.
The values in parentheses are 95% fiducial limits.
Bioassays were also performed with the HD1 isolate of B. thuringiensis subsp. kurstaki (from Dipel; Abbott Laboratories) and B. thuringiensis subsp. aizawai 1857 (Xentari; Abbott Laboratories) transformed with cry1C-t and full-length cry1C constructs (Table 4). For cry1C-t, the construct yielding the highest level of truncated Cry1C, pPFT1Csf-20k, was used, and for full-length cry1C, pPF1C was used. The LC50s of HD-1/pPFT1Csf-20k and HD-1/pPFT1Cs preparations were 7.3 and 3.5 μg/ml, respectively, demonstrating that both strains were more toxic to S. exigua larvae than was the wild-type strain B. thuringiensis subsp. kurstaki HD-1, which had an LC50 of 17.6 μg/ml. However, no significant differences were observed in the LC50s of preparations of B. thuringiensis subsp. aizawai 1857 and the strains harboring cry1C-t (1857/pPFT1Csf-20k) or cry1C (1857/pPFT1Cs).
DISCUSSION
cry1C-t was expressed in this study by using the cyt1A promoter–STAB-SD system (13) in combination with various other genetic elements shown previously to enhance Cry protein synthesis. These included the cry3A transcription terminator (18) and the 20- and 29-kDa so-called helper proteins that improve net synthesis and crystallization. The highest yield of Cry1C-t was obtained with a construct that included the 5′ STAB-SD sequence, the 3′ stem-loop structure, and genes encoding the 20- and 29-kDa helper proteins. Synthesis of Cry1C-t with this construct also resulted in inclusions of Cry1C-t crystals that were 50 to 80% the size of crystals formed by the full-length molecule. These results suggest that use of these enhancer elements in a single expression system might be useful for improving the synthesis of other truncated Cry1 proteins.
Previously, it was shown that the cry1Aa transcription terminator in either orientation at the distal end of the penicillinase (penP) gene or interleukin (IL-2) gene increased the half-life of their mRNAs by 2 to 6 min in Bacillus subtilis and Escherichia coli (24). This resulted in a concomitant increase in PenP and IL-2 synthesis. In contrast to this, in the present study a marked decrease in cry1C-t mRNA resulted when the orientation of the terminator was reversed (Fig. 2). This suggests that variations in cry transcription terminators may be one of the factors that determine the level of Cry synthesis. For example, Cry1C-t inclusions were not obtained with pPFT1C-t and pPFT1Cs-3t(−), constructs which lacked the stem-loop structure and contained the stem-loop structure in a 3′-5′ orientation rather than a 5′-3′ orientation, respectively. Alternatively, Cry1C-t inclusions were obtained with all constructs containing the stem-loop in the same orientation as it occurs naturally in cry3A (18). The lack of synthesis detected when the stem-loop was in the 3′-5′ orientation may have been due to instability of the transcript.
The potential role of the cry11A operon 20-kDa and cry2A operon 29-kDa proteins on the level of synthesis and crystallization of truncated Cry1 proteins has received only limited study. Rang et al. (15) showed that the 20-kDa protein increased net synthesis of Cry1C with deletions in the N-terminal half of the molecule but the proteins contained the C-terminal half of the molecule. Nevertheless, crystalline inclusions of these truncated proteins were not obtained. Similarly, the 20-kDa protein increased net synthesis of Cry2A but did not directly enhance crystallization of this toxin in the absence of the 29-kDa protein (7). In the present study, we showed that when the 20- and 29-kDa protein genes were used independently, though the cry1C-t transcript levels were lower than the levels obtained when these genes were absent (Fig. 2), similar levels of Cry1C-t were produced (Fig. 3). This suggests that the 20- and 29-kDa proteins play no role in cry1C-t expression or mRNA stability but function to enhance net synthesis of Cry1C-t.
The highest transcript level was obtained when both the 20- and 29-kDa protein genes were included in the construct. Thus, it appears that during transcription, cry1C-t was protected from 5′-3′ and 3′-5′ exoribonuclease attack by STAB-SD–29-kDa protein and/or the 20-kDa protein gene fragments that contained 5′ and 3′ stem-loop structures. It is likely that the increase in transcript stability contributed directly to the net increase in the yield of Cry1C-t in cells with pPFT1Csf-20k by about 21% (Fig. 3, lane 7). Moreover, the presence of large inclusions of Cry1C-t synthesized in cells with constructs containing the 20- and 29-kDa protein genes provides further evidence that the helper proteins which these genes encode can function as molecular chaperones, promoting inclusion formation by Cry proteins other than Cry11A and Cry2A.
In previous studies it has been shown that truncated versions of Cry1 proteins are unstable and have little or no toxicity compared to the toxicity of full-length toxins (1, 15, 19, 21). The instability is probably due to the inability of these proteins to fold and crystallize properly. For example, whereas the 20-kDa protein increased net synthesis of truncated Cry1C with deletions in the N-terminal half, no crystalline inclusion were observed (15). This suggests that residues in the N-terminal half of the molecule are essential for crystal formation. Here it is shown that Cry1C-t, which lacked the entire C terminus, was able to form crystalline inclusions, even though these inclusions were approximately 50 to 80% the size of those produced by wild-type Cry1C. In addition, the SDS-PAGE and Western blot data (Fig. 3) showed that Cry1C-t is unstable and is degraded to smaller peptides with molecular masses ranging from 55 to 58 kDa. There was a difference between the molecular mass of Cry1C-t molecules contained in purified crystalline inclusions (68 kDa) and the molecular masses of molecules in the spore-crystal mixture (55 to 58 kDa). This probably resulted from degradation of the 68-kDa Cry1C-t molecules that were not occluded in the inclusions, as these would have been exposed to proteases upon cell lysis (Fig. 5). The smaller 38-kDa peptides (Fig. 5), which were degraded to 31-kDa peptides (Fig. 3B), were possibly the translation products resulting from a second in-frame ATG codon, as noted previously (19).
It has been reported previously that Cry1C-t inclusions produced in E. coli were toxic to Spodoptera littoralis (19). However, this report contained no data on the relative toxicity of Cry1C-t or full-length Cry1C molecules to S. littoralis. The sequence of the Cry1C-t used in the present study was identical to the sequence of the Cry1C-t described previously (19). Here, it is shown that Cry1C-t produced in B. thuringiensis is also toxic to larvae of the beet armyworm, S. exigua, although the toxicity of the truncated protein was 7- to 12.6-fold less than that of the full-length Cry1C (Table 3). The reasons for the lower toxicity of the truncated form are not known at present, but the difference could be due to lower stability and perhaps incorrect folding in the absence of the large C terminus. Despite the lower toxicity of Cry1C-t, synthesis of this molecule in the HD1 isolate of B. thuringiensis subsp. kurstaki, the isolate used in the commercial insecticide Dipel, which lacks Cry1C, increased toxicity to S. exigua as much as fivefold (Table 4). This indicates that the N-terminal half of Cry1C was synthesized effectively in this strain, a finding that may increase this strain's commercial utility. A similar result was not observed with B. thuringiensis subsp. aizawai (Table 3), most likely because this strain already contains Cry1C.
ACKNOWLEDGMENTS
We thank Jeffrey J. Johnson for assistance during the course of this study.
This research was supported in part by grant 96-51 to B.A.F. from the University of California BioSTAR Program.
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